Optimization of the high-performance liquid chromatographic separation of a complex mixture containing urinary steroids, boldenone and bolasterone: application to urine samples

An HPLC separation of a complex mixture containing 13 urinary anabolics and corticoids, and boldenone and bolasterone (synthetic anabolics) has been carried out. The applied optimization method involved the use of binary, ternary and quaternary mobile phases containing acetonitrile, methanol or tetrahydrofuran as organic modifiers. The effect of different reversed-phase packings and temperature on the separation was studied. The optimum separation was achieved by using a water–acetonitrile (60:40, v/v) mobile phase in reversed-phase HPLC at 30°C, allowing the separation of all the analytes in about 24 min. Calibration graphs were obtained using bolasterone or methyltestosterone as internal standards. Detection limits were in the range 0.012–0.107 μg ml−1. The optimized separation was applied to the analysis, after liquid–liquid extraction, of human urine samples spiked with steroids.